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Introduction Barrett’s oesophagus is the condition whereby the normal stratified squamous epithelium at the lower oesophagus is replaced with simple columnar epithelium in the context of gastro-oesophageal reflux disease. This increases the risk of oesophageal adenocarcinoma (OA) ten-fold, and the majority of people diagnosed with OA do not survive for 12 months. Our understanding of the molecular and cellular mechanisms underlying Barrett’s oesophagus is hindered due to lack of at a suitable model. We set out to develop a model from explant tissue biopsies of adult human oesophagus, as opposed to murine models (mice have a keratinised oesophageal epithelium and do not get Barrett’s oesophagus) or the use of cell lines (which do not recapitulate the normal tissue). Method We devised a study protocol and secured ethical approval (REC reference number 13/YH/0197) to obtain forceps biopsy specimens from consenting adults attending for routine diagnostic gastroscopies at the Royal United Hospital Bath. Specimens were then transported to the University of Bath where we have tested multiple culture conditions. These include choice of culture media, substrate (eg glass, plastic – coated or uncoated, semi-permeable membrane at air/liquid interface), cell adhesion factors (eg collagen I, collagen IV, fibronectin, Matrigel®), choice of tissue preparation (enzyme treatment with dispase in the absence or presence of trypsin, dissection with scissors or minced with a blade), plating density, media volume and use of a cell feeder layer. Results We have recruited 42 participants to date and attempted tissue culture on 36 of these. (Biopsies from 6 patients have been processed for RNA analysis.) Over time we have gradually optimised culture conditions. Of the most recent 6 participants, 5 have cultured successfully with cell survival for at least 14 days. Explant colony growth had been particularly vigorous (>12 mm) when plated on Matrigel® and a feeder layer cell line (mitomycin C-inactivated mouse embryonic fibroblast (iMEF) cell line 3T3-Swiss albino, ATCC® CCL-92™) on plastic coverslips. Explants have also adhered and cultured successfully on collagen IV-coated (sc-29010, Santa Cruz Biotech) plastic coverslips without a feeder layer. Explant colonies were fixed/permeabalised before immunofluorescent staining was performed for cytokeratin 14 (K14), smooth muscle actin (SMA), p63, E-cadherin and SLUG antigens. iMEFs are clearly delineated with SMA staining and the explant outgrowths are positive for K14, p63, E-cadherin and SLUG. Conclusion It is possible to culture adult human oesophageal tissue in vitro from standard pinch-biopsy forceps specimens at gastroscopy and we are in the final stages of optimising conditions, confirming characterisation by repeating experiments and devising a protocol. Disclosure of interest None Declared. |
